Synthesis and structure of two novel trans-platinum complexes

The synthesis and structural characterization (XRD and NMR) of two new trans-platinum complexes are presented.


Introduction
Platinum-based drugs have been used for chemotherapeutic treatment of cancer since 1965, when Rosenberg's group discovered the cytotoxic activity of cisplatin Pt(NH 3 ) 2 Cl 2 (Rosenberg et al., 1965). In 1970, cisplatin was approved for application in testicular and ovarian cancer by the US Food and Drug Administration and in several European countries (Wiltshaw, 1979). It is prescribed also for the treatment of a wide array of other tumors such us head-and-neck, esophagus, stomach, colon, bladder, cervix, pancreas, liver, kidney and prostate cancers. Although cisplatin has been used for more than 40 years, the severe side effects and the drug resistance of many cancer types have been the major limitations for its clinical application (Lippert, 1999;Giaccone, 2000;Oberoi et al., 2013). Cisplatin resistance may be associated with reduced drug uptake, enhanced efflux, intracellular detoxification by glutathione, increased DNA repair, decreased mismatch repair, defective apoptosis, modulation of signaling pathways or the presence of quiescent non-cycling cells (Steward, 2007;Rabik & Dolan, 2007;Kuo et al., 2007;Boulikas et al., 2007;Heffeter et al., 2008;Reedijk, 2011). In the last 45 years, although a great effort has been made for synthesizing platinum compounds with reduced side effects and propensity to induce drug resistance (Jakupec et al., 2003), none of them has reached worldwide clinical application. Only five complexes (carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin) have been registered for clinical treatment with regional approval (cisplatin, carboplatin and oxaliplatin are FDA-approved, nedaplatin in Japan and lobaplatin in China).
According to early structure-property relationship studies, only platinum compounds with cis-configuration exhibit antitumor activity (Kelland, 2007). In recent decades, however, it has been observed also that many trans-platinum(II) complexes exhibit anticancer activity comparable with the cisisomer and cisplatin Montero et al., 1999;Kasparkova et al., 2003a,b). The promising biological activity of trans-platinum(II) complexes encouraged us to synthesize and characterize a novel trans-platinum(II) complex with a benzamide ligand, namely trans-{PtCl 2 [HN C(OH)C 6 H 5 ] 2 }. Derivatives of benzamide are known to possess cytotoxic activity (Vernhet et al., 1997;Rauko et al., 2001;Zhang et al., 2022) and such a ligand is interesting also owing to the occurrence of a hydroxyl group which can serve as a hydrogen-bond donor or acceptor.
Although the platinum drugs currently used for cancer treatment consist of platinum cations with oxidation state +2, in recent years platinum(IV) species have also been investigated. The interest in Pt IV complexes arises from their greater inertness to ligands substitution compared with Pt II counterparts, a feature that allows chemical modification of the ligands without breaking the metal-ligand bond. The slow exchange rate of ligands coordinated to Pt IV plays an increasingly important role for the development of new nanotechnology for delivering platinum drugs to cancer cells (Dhar et al., 2011;Min et al., 2010). The presence of two extra coordination sites can also be used in combination with other drugs, or for modifying biological targets other than DNA in the cell. In addition, platinum(IV) complexes are stable in the oxidizing extracellular environment and they can easily reach the platinum(II) oxidation state inside the cell (Wong & Giandomenico, 1999). The increasing interest for Pt IV species prompted us to extend our investigation to a platinum(IV) complex, trans-[PtCl 4 (NH 3 ){HN C(OH) t Bu}]. The bulky substituent (tertiary butyl group) in the amide ligand could potentiate the cellular uptake of the complex via passive diffusion through the cell membrane, because of the greater affinity for lipophilic environments, while the hydroxyl group would preserve the water solubility.

trans-[PtCl 2 {HN C(OH)C 6 H 5 } 2 ] synthesis
Compound 1 was prepared by protonation of the K 2 {trans-[Pt II Cl 2 (H 2 NC( O)C 6 H 5 ) 2 ]} salt: the reactant (0.6 g, 1 mmol) was dissolved in ice-cold water and treated with an excess of hydrochloric acid (10 ml, 6 M). The yellow precipitate separated from the solution was collected by filtration of the mother liquor, washed with ice cold water and dried in a stream of dry air. Compound 1 was isolated, crystallized in chloroform giving yellow lamellar crystals ( Fig. 1) and then characterized using NMR spectroscopy and X-ray diffraction.

trans-[PtCl 4 (NH 3 ){HN C(OH) t Bu}] synthesis
The trans-[Pt II Cl 2 (NH 3 )(NC t Bu)] precursor [0.1780 g, 0.49 mmol, M r (molecular weight) = 366 g mol À1 ] was suspended in chloroform (30 ml) and Cl 2 (2 ml), and stirred at 293 K for 30 min. The resulting solution was taken to dryness under reduced pressure, giving a yellow precipitate of trans-[Pt IV Cl 4 (NH 3 )(NC t Bu)]. The obtained complex was treated with KOH, then neutralized with HCl. The complex was isolated, crystallized in a mixture of chloroform/pentane, and characterized by NMR spectroscopy followed by X-ray diffraction.

X-ray single crystal determination
Reflections were collected on a Bruker AXS X8 APEX CCD diffractometer equipped with a four-circle Kappa goniometer and a 4K CCD detector (Mo K radiation). Data reduction and unit-cell refinement were carried out with the SAINT package (Bruker, 2003). The reflections were indexed, integrated and corrected for Lorentz, polarization and absorption effects with the program SADABS (Sheldrick, 2010). All calculations and molecular graphics were carried out using SIR92 (Altomare et al., 1993), PARST97 (Nardelli, 1995), WinGX (Farrugia, 1999), CRYSTALS (Carruthers et al., 2003), MERCURY (Macrae et al., 2020) and ORTEP-3 for Windows packages (Farrugia, 2012). Details of the experiment and crystal data are given in Table 1. Selected bond lengths and angles are listed in Table 2.
The unit-cell parameters were calculated from all reflections. Anisotropic displacement parameters (ADPs) for hydrogen and all non-hydrogen atoms were refined isotropically and anisotropically, respectively. The crystal structure was solved using direct methods in space groups P1 and P2 1 for compounds 1 and 2, respectively, and the models were refined using full-matrix least-squares.
2.3.1. Compound 1. The difference Fourier synthesis shows one maximum at the midpoint of two oxygen positions with the refinement resulting in grossly anisotropic displacement parameters corresponding to a 'cigar-shaped' ellipsoid. The disorder is refined using the so-called split-atom model strategy. The two partial atoms are refined independently, even with the sum of their site occupancies constrained to unity (Fig. 2). The hydrogen atoms were located by Fourier difference except for the hydrogen atoms located on oxygen sites which were placed at calculated positions. All hydrogen atoms were refined isotropically.
2.3.2. Compound 2. The asymmetric unit includes two disordered complexes: the disorder of the first complex (a) involves the methyl in the tertiary butyl group [-C(CH 3 ) 3 ] and the oxygen atom in the amide moiety (NCO). The split-atom model has been used to model the disorder (Fig. 3). Since the hydrogen atoms bound to the split oxygen atoms have not been found by Fourier difference, they were added manually and refined isotropically. In the second complex (b) in the asymmetric unit of compound 2 shows an electron density symmetrically distributed around a a local plane through the N2 and C2 atoms of the pivaloamide group. The disorder was modeled by splitting the C1 carbon atom and the tertiary butyl group (Fig. 4).  Computer programs: COLLECT (Nonius, 2001), DENZO/SCALEPACK (Otwinowski & Minor, 1997), CrysAlis (Oxford Diffraction, 2002), SUPERFLIP (Palatinus & Chapuis, 2007), CRYSTALS (Betteridge et al., 2003), CAMERON (Watkin et al., 1996).

Figure 3
Oxygen and methyl splitting in compound 2a. Color coding for atoms: red: oxygen; dark gray: carbon; violet: nitrogen; light grey: platinum; green: chloride.

Results and discussion
3.1. NMR spectroscopy 3.1.1. Compound 1: platinum(II). The H 1 -NMR spectrum was recorded on a Bruker Avance DPX 300 MHz WB instrument at 295 K in acetone-d 6 . 1 H chemical shifts were referenced to TMS by using the residual protic peak of acetone-d 6 as internal reference. The 1 H-NMR spectrum (Fig. 5) shows two broad signals at $11.08 ppm and $8.31 ppm assigned to the OH and NH protons in the amide ligand, respectively, and three aromatic proton contributions at 7.96 ppm, 7.68 ppm and 7.58 ppm from the ortho, para and meta protons, respectively.
3.1.2. Compound 2: platinum(IV). 1 H-NMR spectrum was collected at 295 K in CDCl 3 . 1 H chemical shifts were referenced to TMS by using the residual protic peak of CDCl 3 as internal reference. The 1 H-NMR spectrum (Fig. 6) (2022)}. Moreover, two single proton resonances at $6.90 and 10.25 can be assigned to the NH and OH groups in the amide moiety. It is worth noting that the shape of the hydroxyl proton peak depends upon the nature of R [-C(CH 3 ) 3 in compound 2 or -C 6 H 5 in compound 1]: the signal is sharp for the t Bu derivative and broader for the phenyl group. This feature may be explained by the chemical exchange process involving the hydroxyl proton and water impurities. The exchange rate is expected to increase with the acidity of the hydroxyl proton.  Table 2 Selected bond lengths and angles (Å , ) for compound 1 and 2.
The hydroxyl group points toward the chloride ligand resulting in an intramolecular hydrogen bond that stabilizes the complex (Fig. 7).
The molecular crystal packing is mainly governed by Á Á Á stacking interactions and van der Waals intermolecular forces, involving the benzene ring of adjacent molecules with an intermolecular distance of $3.62 Å (Sinnokrot & Sherrill, 2006). The van der Waals intermolecular interactions involve hydrogen of the benzene ring and oxygen atom of the hydroxyl group [C7Á Á ÁO1A 3.280 (3) Å , H31Á Á ÁO2 2.613 (1) Å , C3-H31Á Á ÁO2 146.03 (8) ], resulting in the crystal packing shown in Fig. 8. 3.2.2. Compound 2: platinum(IV). The crystal structure features two enantiomers in the cell [Flack parameter = 0.47 (1)]. The Pt IV atom has an octahedral coordination geometry with four chloride ligands and two nitrogen atoms (with hybridization sp 2 and sp 3 for amide and ammine ligands, respectively) in trans configuration (Fig. 9). The bond distances between platinum and ligands (Pt-N and Pt-Cl distances in Table 2    previously for compound 1 and in the literature for platinum(III) and platinum(II) complexes (Vinci & Chateigner, 2022;Fabijań ska et al., 2015;Grabner & Bukovec, 2015;Cini et al., 1999;. In the amide ligand, the C-N and C-O distances average 1.20 (7) Å and 1.13 (1) Å , respectively, due to the double bond delocalization over the N-C-O moiety as observed also for compound 1. The C6-N4-Pt2, Pt1-N2-C11 and Pt1-N2-C10 angles are 132.7 (5) , 140.2 (5) and 141.3 (4) , respectively, similar to the angle found for compound 1. The larger angle is probably due to intramolecular hydrogen bonds involving the hydroxyl hydrogen and the chloride ligand (Fig. 10). The bond angles within the coordination sphere deviate significantly from the ideal value of 90 . For instance, in compound 2 with the atom labeled Pt2 (Fig. 9), two angles are particularly large [93.67 (19) and 93.60 (17) ] for N4-Pt2-Cl5 and N4-Pt2-Cl8 angles, respectively) and two are particularly small [87.57 (16) and 86.60 (19) ] for N3-Pt2-Cl6 and N3-Pt2-Cl8 angles, respectively). This feature is due to the hydrogen bond interactions between the hydroxyl group and the chloride ligands. The same behavior has been observed in the second enantiomer in the unit cell.
The molecular packaging (Fig. 11) is governed by hydrogen bonds and van der Waals interactions (Table 3). The intermolecular hydrogen bonds are observed between amide and chloride ligands. The intermolecular van der Waals interactions occur between the tert-butyl groups and the chloride ligands.

Conclusions
For compound 1 we observed that the protonation of K 2 {trans-[PtCl 2 (H 2 NC( O)C 6 H 5 ) 2 ]} salt in ice-cold water, treated with an excess of hydrochloric acid, gives the new trans-[PtCl 2 {HN C(OH)C 6 H 5 } 2 ] complex stable at room temperature. Spectroscopic studies indicate that the benzamide ligand is present in compound 1. The X-ray structural determination confirmed that the central platinum(II) atom is four-coordinated via two nitrogen atoms of the benzamide ligands and two chloride anions. The dihedral angle between PtCl 2 N 2 and the benzene ring plane is 21 (1)  ORTEP diagram of two enantiomers of compound 2. The ellipsoids enclose 30% probability. Color coding for atoms: white: hydrogen; green: chlorine; blue: nitrogen; gray: carbon; red: oxygen.

Figure 11
ORTEP drawing of packing of compound 2 governed by intermolecular hydrogen bonds and van der Waals interactions. The ellipsoids enclose 30% probability. Atom color coding: white for hydrogen, green for chlorine, blue for nitrogen, gray for carbon and red for oxygen. lattice framework is governed by Á Á Á and van der Waals intermolecular interactions.
Compound 2 was prepared by neutralization with HCl of a trans-[PtCl 4 (NH 3 )(NC t Bu)] solution in KOH. As expected, the X-ray structure contains a platinum(IV) atom six-coordinated by an ammine, four chloride and a pivalamide ligands with trans configuration. NMR spectroscopy confirmed the presence of the pivalamide ligand and the octahedral geometry.
A common feature between these two structures is the occurrence of intramolecular hydrogen bonds between the hydroxyl group and the chloride ligand. The next step in this work is the evaluation of the cytotoxic effect of these new platinum compounds against human and murine cancer cell lines, as well as the toxicity towards healthy cells and these effects will be compared with those of other cisplatin compounds. Table 3 Intra and intermolecular hydrogen bonds, and intermolecular van der Waals interactions (Å , ) for compound 2.